Breast cancer brain metastasis, which occurs in more than one third of metastatic patients with HER2- positive tumors, is extremely difficult to treat and has a typical survival rate of < 1 year. Although aberrant HER2 receptor overexpression in breast cancer is highly correlated with brain metastasis, molecularly-targeted anti-HER2 therapy, such as trastuzumab and lapatinib, poorly cross the blood brain barrier (BBB) to reach the metastatic site at therapeutic concentrations. This proposal seek to overcome a critical barrier for delivery of the molecularly-targeted anti-HER2 drug, lapatinib, t a specific metastatic site - the brain. Our approach exploits the unique properties of a brain homing peptide, Angiopep-2, to improve penetration of lapatinib across the BBB in order to treat HER2+ brain metastasis. The 19-amino acid peptide, Angiopep-2, binds to the low-density lipoprotein receptor-related protein (LRP-1) receptor and is transcytosed into the brain with more efficiency than transferrin. The goal of this research is to develop brain-permeable lapatinib Angiopep-2 conjugates that can enhance therapeutic drug levels in the brain to treat HER2+ breast cancer brain metastasis. We hypothesize that lapatinib-Angiopep-2 conjugates will cross the BBB more efficiently than lapatinib itself and lead to higher lapatinib accumulation in the brain metastasis. Consequently, therapeutic benefits will be obtained in targeting and destroying HER2+ breast cancer brain metastasis. We plan to test the hypothesis through three specific aims: (1) optimize lapatinib-Angiopep conjugates with improved solubility, serum stability, and ability to achieve localized lapatinib release; (2) demonstrate the effectiveness of brain penetration, cellular uptake and release, and cytotoxic potency of the optimized conjugates; (3) validate enhanced brain accumulation and therapeutic efficacy of optimized conjugates in HER2-overexpressing breast cancer brain metastasis models in immunodeficient mice. We will optimize synthetic protocols to chemically attach lapatinib to the peptide backbone of Angiopep-2 via different cross-linkers. We will study the stability of conjugates in mouse and human serum and cytotoxicity in HER2-overexpressing breast cancer cells. We will determine BBB transport rates of the conjugates using an in situ mouse brain perfusion technique. We will test the efficacy of conjugates in our established breast cancer brain metastasis mouse models by intracardiac and intracranial injection of brain-seeking HER2- overexpressing human breast cancer cells into nude mice, and will monitor tumor growth and/or regression after conjugate treatment. Successful completion of this project will identify a lapatinib delivery system that is able to cross the BBB and is active against breast cancer brain metastases animal models. If successful, the research can be translatable into the clinic and will potentially lead to a major step forward in extending the lives of breast cancer patients with HER2-positive brain metastases. This work will have a broader impact upon the delivery of other molecularly-targeted tyrosine kinase inhibitors, such as gefitinib and erlotinib, which are structural analogs of lapatinib, for the treatment of CNS metastases originating from other cancers.